COMPOSITIONS PHARMACEUTIQUES DE MICROCIONA PROLIFERA D'EPONGE MARINE

PHARMACEUTICAL COMPOSITIONS OF MARINE SPONGE MICROCIONA PROLIFERA

Abrégé français

La présente invention concerne des compositions et des procédés pour traiter une resténose et des maladies auto-immunes. Ces procédés consistent à administrer une composition qui comprend des polypeptides, des glucides et/ou des extraits et leur analogues, dérivés et conjugués, issus de Microciona prolifera d'éponge marine.

Abrégé anglais

The present invention provides compositions and methods for treating
restenosis and autoimmune diseases. The methods provided herein include the
administration of a composition comprising of the class of polypeptides,
carbohydrates and/or extracts and their analogues, derivatives and conjugates,
originating from the marine sponge Microciona prolifera.

Revendications

WE CLAIM:
1. A polypeptide comprising an amino acid sequence selected from a group
consisting of B-2
(SEQ ID NO: 2), B-3 (SEQ ID NO 3), B-6 (SEQ ID NO 4), B-9 (SEQ ID NO: 5), B-10
(SEQ ID NO: 6), and B-11 (SEQ ID NO: 7).
2. A pharmaceutical composition comprising a polypeptide of claim 1.
3. A pharmaceutical composition for the treatment of restenosis comprising:
a. at least one polypeptide comprising an amino acid sequence selected from a
group
consisting of B-2 (SEQ ID NO: 2), B-3 (SEQ ID NO 3), B-6 (SEQ ID NO 4), B-9
(SEQ ID NO: 5), B-10 (SEQ ID NO: 6), B-11 (SEQ ID NO: 7), and analogues
and derivatives thereof; and
b. a pharmaceutically acceptable carrier.
4. A pharmaceutical composition for the treatment of an autoimmune disease,
comprising:
a. at least one polypeptide comprising an amino acid sequence selected from a
group
consisting of B-2 (SEQ ID NO: 2), B-3 (SEQ ID NO 3), B-6 (SEQ ID NO 4), B-
9 (SEQ ID NO: 5), B-10 (SEQ ID NO: 6), B-11 (SEQ ID NO: 7), and analogues
and derivatives thereof; and
b. a pharmaceutically acceptable carrier.
5. The pharmaceutical composition of claim 4, wherein the autoimmune disease
is multiple
sclerosis.
6. The pharmaceutical composition of claim 4, wherein the autoimmune disease
is diabetes.
7. The pharmaceutical composition of claim 4, wherein the autoimmune disease
is an
inflammatory joint disease.
27

8. The pharmaceutical composition of claim 7, wherein the inflammatory joint
disease is
arthritis.
9. A pharmaceutical composition for the treatment of multiple sclerosis,
comprising:
a. at least one polypeptide comprising an amino acid sequence selected from a
group
consisting of B-2 (SEQ ID NO: 2), B-3 (SEQ ID NO 3), B-6 (SEQ ID NO 4), B-
9 (SEQ ID NO: 5), B-10 (SEQ ID NO: 6), B-11 (SEQ ID NO: 7), and analogues
and derivatives thereof; and
b. a pharmaceutically acceptable carrier.
10. An antibody which binds to a polypeptide comprising an amino acid sequence
selected
from a group consisting of B-1 (SEQ ID NO: 1), B-2 (SEQ ID NO: 2), B-3 (SEQ ID
NO
3), B-6 (SEQ ID NO 4), B-9 (SEQ ID NO: 5), B-10 (SEQ ID NO: 6), B-11 (SEQ ID
NO:
7), and analogues and derivatives thereof.
11. An antibody according to claim 10, wherein the antibody is a human
monoclonal
antibody.
12. An antibody according to claim 10, wherein the antibody is a Fab fragment
of an
antibody.
13. A vaccine composition for the treatment of a disease selected from a group
consisting of
autoimmune diseases and restenosis comprising an antigen for the antibody of
claim 10.
14. A method for treating restenosis comprising a step of administering to a
patient at least
one compound selected from a group consisting of (a) a polypeptide comprising
an amino
acid sequence selected from a group consisting of B-1 (SEQ ID NO: 1), B-2 (SEQ
ID NO:
2), B-3 (SEQ ID NO 3), B-6 (SEQ ID NO 4), B-9 (SEQ ID NO: 5), B-10 (SEQ ID NO:
6), B-11 (SEQ ID NO: 7), and analogues and derivatives thereof; and (b) an
antibody to
the polypeptide of (a).
15. A method for treating an autoimmune disease comprising a step of
administering to a
patient at least one compound selected from a group consisting of (a) a
polypeptide
28

comprising an amino acid sequence selected from a group consisting of B-1 (SEQ
ID NO:
1), B-2 (SEQ ID NO: 2), B-3 (SEQ ID NO 3), B-6 (SEQ ID NO 4), B-9 (SEQ ID NO:
5),
B-10 (SEQ ID NO: 6), B-11 (SEQ ID NO. 7), and analogues and derivatives
thereof; and
(b) an antibody to the polypeptide of (a).
16. The method of claim 15 wherein the autoimmune disease is multiple
sclerosis.
17. The method of claim 15 wherein the autoimmune disease is diabetes.
18. The method of claim 15 wherein the autoimmune disease is an inflammatory
joint disease.
19. The method of claim 18 wherein the inflammatory joint disease is
arthritis.
20. A method for treating multiple sclerosis comprising a step of
administering to a patient at
least one compound selected from a group consisting of (a) a polypeptide
comprising an
amino acid sequence selected from a group consisting of B-1 (SEQ ID NO: 1), B-
2 (SEQ
ID NO: 2), B-3 (SEQ ID NO 3), B-6 (SEQ ID NO 4), B-9 (SEQ ID NO: 5), B-10 (SEQ
ID NO: 6), B-11 (SEQ ID NO: 7), and analogues and derivatives thereof; and (b)
an
antibody to the polypeptide of (a).
21. A method for treating restenosis comprising a step of administering to a
patient at least
one compound selected from a group consisting of:
a. GlcNAc.beta.1-3Fuc;
b. Gal(31-4GlcNAc(31-3Fuc;
c. (4,6-Pyr)Gal.beta.1-4GlcNAc.beta.1-3Fuc;
d. GlcNAc.beta.1-3Fuc
¦
3SO3; and
e. analogues and derivatives thereof.
29

22. A method for treating an autoimmune disease comprising a step of
administering to a
patient at least one compound selected from a group consisting of:
a. GlcNAc.beta.1-3Fuc;
b. Gal.beta.1-4GlcNAc.beta.1-3Fuc;
c. (4,6-Pyr)Gal.beta.1-4GlcNAc.beta.1-3Fuc;
d. GlcNAc.beta.1-3Fuc
3503; and
e. analogues and derivatives thereof.
23. The method of claim 22 wherein the autoimmune disease is multiple
sclerosis.
24. The method of claim 22 wherein the autoimmune disease is diabetes.
25. The method of claim 22 wherein the autoimmune disease is an inflammatory
joint disease.
26. The method of claim 2S wherein the inflammatory joint disease is
arthritis.
27. A method for treating multiple sclerosis comprising a step of
administering to a patient at
least one compound selected from a group consisting of:
a. GlcNAc.beta.1-3Fuc;
b. Gal.beta.1-4GlcNAc.beta.1-3Fuc;
c. (4,6-Pyr)Gal.beta.1-4GlcNAc.beta.1-3Fuc;
d. GlcNAc.beta.1-3Fuc
35SO3; and

e. analogues and derivatives thereof.
28. A method for treating restenosis comprising a step of administering to a
patient a
pharmaceutical composition comprising a purified extract from Microciona
prolifera.
29, A method for treating an autoimmune disease comprising a step of
administering to a
patient a pharmaceutical composition comprising a purified extract from
Microciona
prolifera.
30. The method of Claim 29 wherein the autoimmune disease is multiple
sclerosis.
31. The method of Claim 29 wherein the autoimmune disease is diabetes.
32. The method of Claim 29 wherein the autoimmune disease is an inflammatory
joint
disease.
33. The method of Claim 32 wherein the inflammatory joint disease is
arthritis.
34. A method for treating multiple sclerosis comprising a step of
administering to a patient a
pharmaceutical composition comprising a purified extract from Microciona
prolifera.
35. A method for treating restenosis comprising a step of administering to a
patient at least
one compound selected from a group consisting of (a) a polypeptide comprising
an amino
acid sequence selected from a group consisting of B-1(SEQ ID NO: 1), B-2 (SEQ
ID NO:
2), B-3 (SEQ ID NO 3), B-6 (SEQ ID NO 4), B-9 (SEQ ID NO: 5), B-10 (SEQ ID NO:
6), B-11 (SEQ ID NO: 7), and analogues and derivatives thereof; and (b) an
antibody to
the polypeptide of (a).
36. A method for heating an autoimmune disease comprising a step of
administering to a
patient at least one compound selected from a group consisting of (a) a
polypeptide
comprising an amino acid sequence selected from a group consisting of B-1(SEQ
ID NO:
1), B-2 (SEQ ID NO: 2), B-3 (SEQ ID NO 3), B-6 (SEQ ID NO 4), B-9 (SEQ ID NO:
5), B-10 (SEQ ID NO: 6), B-11 (SEQ ID NO: 7), and analogues and derivatives
thereof;
and (b) an antibody to the polypeptide of (a).
31

37. The method of claim 36, wherein the autoimmune disease is multiple
sclerosis.
38. The method of claim 36, wherein the autoimmune disease is diabetes.
39. The method of claim 36, wherein the autoimmune disease is an inflammatory
joint
disease.
40. The method of claim 39, wherein the inflammatory joint disease is
arthritis.
41. A method according to any of claims 14 - 40, wherein a dose is
administered according to
a regime selected from a group consisting of a single dose, multiple daily
doses, multiple
weekly doses and multiple monthly doses.
42. Use of at least one polypeptide comprising an amino acid sequence selected
from a group
consisting of B-1 (SEQ ID NO: 1), B-2 (SEQ ID NO: 2), B-3 (SEQ ID NO 3), B-6
(SEQ
ID NO 4), B-9 (SEQ ID NO: 5), B-10 (SEQ ID NO: 6), B-11 (SEQ ID NO: 7), and
analogues and derivatives thereof for the treatment of restenosis.
43. Use of at least one polypeptide comprising an amino acid sequence selected
from a group
consisting of B-1 (SEQ ID NO: 1), B-2 (SEQ ID NO: 2), B-3 (SEQ ID NO 3), B-6
(SEQ
ID NO 4), B-9 (SEQ ID NO: 5), B-10 (SEQ ID NO: 6), B-11 (SEQ ID NO: 7), and
analogues and derivatives thereof for the treatment of an autoimmune disease.
44. The use according to claim 43, wherein the autoimmune disease is selected
from a group
consisting of multiple sclerosis, diabetes, and an inflammatory joint disease.
45. The use according to claim 44, wherein the inflammatory joint disease is
arthritis.
46. Use of at least one polypeptide comprising an amino acid sequence selected
from a group
consisting of B-1 (SEQ ID NO: 1), B-2 (SEQ ID NO: 2), B-3 (SEQ ID NO 3), B-6
(SEQ
ID NO 4), B-9 (SEQ ID NO: 5), B-10 (SEQ ID NO: 6), B-11 (SEQ ID NO: 7), and
analogues and derivatives thereof for the treatment of multiple sclerosis.
47. Use of a compound selected from a group consisting of:
32

a. GlcNAc.beta.1-3Fuc;
b. Gal.beta.1-4GlcNAc.beta.1-3Fuc;
c. (4,6-Pyr)Gal.beta.1-4GlcNAc.beta.1-3Fuc;
<IMG>
e, analogues and derivatives thereof
for the treatment of restenosis.
48. Use of a compound selected from a group consisting of:
a. GlcNAc.beta.1-3Fuc;
b. Gal.beta.1-4GlcNAc.beta.1-3Fuc;
c. (4,6-Pyr)Gal.beta.1-4GlcNAc.beta.1-3Fuc;
<IMG> and
e. analogues and derivatives thereof
for the treatment of an autoimmune disease.
49. The use according to claim 48, wherein the autoimmune disease is selected
from a group
consisting of multiple sclerosis, diabetes, and an inflammatory joint disease.
50. The use according to claim 49 wherein the inflammatory joint disease is
arthritis.
33

51. Use of a compound selected from a group consisting of:
a. GlcNAc.beta.1-3Fuc;
b. Gal.beta.1-4GlcNAc.beta.1-3Fuc;
c. (4,6-Pyr)Gal.beta.1-4GlcNAc.beta.1-3Fuc;
<IMG> and
e. analogues and derivatives thereof
for the treatment of multiple sclerosis
52. Use of a pharmaceutical composition comprising a purified extract from
Microciona
prolifera for the treatment of restenosis.
53. Use of a pharmaceutical composition comprising a purified extract from
Microciona
prolifera for the treatment of an autoimmune disease.
54. The use of claim 53, wherein the autoimmune disease is selected from a
group consisting
of multiple sclerosis, diabetes, and an inflammatory joint disease.
55. The use of claim 54 wherein the inflammatory joint disease is arthritis.
56. Use of a pharmaceutical composition comprising a purified extract from
Microciona
prolifera for the treatment of multiple sclerosis.
57. Use of an antibody which binds to a polypeptide comprising an amino acid
sequence
selected from a group consisting of B-1(SEQ ID NO: 1), B-2 (SEQ ID NO: 2), B-3
(SEQ
ID NO 3), B-6 (SEQ ID NO 4), B-9 (SEQ ID NO: 7), B-10 (SEQ ID NO: 6), B-11
(SEQ
ID NO: 7), and analogues and derivatives thereof for the treatment of
restenosis.
34

58. Use of an antibody which binds to a polypeptide comprising an amino acid
sequence
selected from a group consisting B-1(SEQ ID NO: 1), B-2 (SEQ ID NO: 2), B-3
(SEQ ID
NO 3), B-6 (SEQ ID NO 4), B-9 (SEQ ID NO: 5), B-10 (SEQ ID NO: 6), and B-11
(SEQ ID NO: 7) for the treatment of an autoimmune disease.
59. The use of claim 58, wherein the autoimmune disease is selected from a
group consisting
of multiple sclerosis, diabetes, and an inflammatory joint disease.
60. The use of claim 59, wherein the inflammatory joint disease is arthritis.
61. Use of an antibody which binds to a polypeptide comprising an amino acid
sequence
selected from a group consisting of B-1(SEQ ID NO: 1), B-2 (SEQ ID NO: 2), B-3
(SEQ
ID NO 3), B-6 (SEQ ID NO 4), B-9 (SEQ ID NO: 7), B-10 (SEQ ID NO: 6), B-11
(SEQ
ID NO: 7), and analogues and derivatives thereof for multiple sclerosis.
62. A pharmaceutical composition for the treatment of restenosis, comprising:
a. a purified extract from Microciona prolifera having a peak at about 2.7kDa
using
MALDI spectroscopy; and
b. a pharmaceutically acceptable carrier.
63. A pharmaceutical composition for the treatment of an autoimmune disease,
comprising:
a. a purified extract from Microciona prolifera having a peak at about 2.7kDa
using
MALDI spectroscopy; and
b. a pharmaceutically acceptable carrier.
64. The pharmaceutical composition of claim 63, wherein the autoimmune disease
is selected
from a group consisting of multiple sclerosis, diabetes, and an inflammatory
joint disease.
65. The pharmaceutical composition of claim 64 wherein the inflammatory joint
disease is
arthritis.
35

66. A pharmaceutical composition for the treatment of restenosis comprising,
an extract from
Microciona prolifera obtained by
a. washing one or more cuttings of Microciona prolifera in a solution of cold
calcium
and magnesium free artificial seawater (CMFASW), wherein said cuttings contain
one or more MAF-containing cells and a fibro-silacaceous support;
b. rotating the cuttings in CMFASW;
c. separating the MAF-containing cells from the fibro-silacaceous support;
d. incubating and rotating the MAF-containing cells;
e. centrifuging the MAF-containing cells and retaining the supernatant;
f. adding EDTA and optionally a preservative to obtain a supernatant solution;
g, storing the supernatant solution at about 4 degrees for at least 120 hours;
h. dialyzing the supernatant solution to obtain a retenate; and
i. lyophilizing the retenate to obtain the extract.
67. A pharmaceutical composition for the treatment of an autoimmune disease
comprising, an
extract from Microciona prolifera obtained by
a. washing one or more cuttings of Microciona prolifera in a solution of cold
calcium
and magnesium free artificial seawater (CMFASW), wherein said cuttings contain
one or more MAF-containing cells and a fibro-silacaceous support;
b, rotating the cuttings in CMFASW;
c. separating the MAF-containing cells from the fibro-silacaceous support;
d. incubating and rotating the MAF-containing cells;
36

e. centrifuging the MAF-containing cells and retaining the supernatant;
f. adding EDTA and optionally a preservative to obtain a supernatant solution;
g. storing the supernatant solution at about 4 degrees for at least 120 hours;
h. dialyzing the supernatant solution to obtain a retenate; and
i. lyophilizing the retenate to obtain the extract.
68. The pharmaceutical composition of claim 67 wherein the autoimmune disease
is selected
from a group consisting of multiple sclerosis, diabetes, and an inflammatory
joint disease.
69. The pharmaceutical composition of claim 68 wherein the inflammatory joint
disease is
arthritis.
70. A pharmaceutical composition for the treatment of multiple sclerosis
comprising, an
extract from Microciona prolifera obtained by
a. washing one or more cuttings of Microciona prolifera in a solution of cold
calcium
and magnesium free artificial seawater (CMFASW), wherein said cuttings contain
one or more MAF-containing cells and a fibro-silacaceous support;
b. rotating the cuttings in CMFASW;
c. separating the MAF-containing cells from the fibro-silacaceous support;
d. incubating and rotating the MAF-containing cells;
e, centrifuging the MAF-containing cells and retaining the supernatant;
f. adding EDTA and optionally a preservative to obtain a supernatant solution;
g. storing the supernatant solution at about 4 degrees for at least 120 hours;
h. dialyzing the supernatant solution to obtain a retenate; and
37

i. lyophilizing the retenate to obtain the extract.
71. Use of a polypeptide comprising an amino acid sequence selected from a
group consisting
of B-1 (SEQ ID NO: 1), B-2 (SEQ ID NO: 2), B-3 (SEQ ID NO 3), B-6 (SEQ ID NO
4),
B-9 (SEQ ID NO: 5), B-10 (SEQ ID NO: 6), B-11 (SEQ ID NO: 7) and analogues and
derivatives thereof to identify ligands on a cell surface that may serve as
potential
therapeutic targets.
72. Use of a compound selected from a group consisting of:
a. GlcNAc.beta.1-3Fuc;
b. Gal.beta.1-4GlcNAc.beta.1-3Fuc;
c. (4,6-Pyr)Gal.beta.1-4GlcNAc.beta.1-3Fuc;
<IMG>
e. analogues and derivatives thereof
to identify at least one ligand on a cell surface that may serve as a
potential therapeutic
target.
73. Use of a purified extract from Microciona prolifera to identify at least
one ligand on a cell
surface that may serve as a potential therapeutic target.
74. A therapeutic target, comprising at least one ligand on a cell surface
which binds to a
compound selected from a group consisting of B-1 (SEQ ID NO: 1) B-2 (SEQ ID
NO: 2)
B-3 (SEQ ID NO 3), B-6 (SEQ ID NO 4), B-9 (SEQ ID NO: 5), B-10 (SEQ ID NO: 6),
B-
11 (SEQ ID NO: 7) and analogues and derivatives thereof.
75. A therapeutic target, comprising at least one ligand on a cell surface
which binds to a
compound selected from a group consisting of
38

a. GlcNAc.beta.1-3Fuc;
b. Gal.beta.1-4GlcNAc.beta.1-3Fuc;
c. (4,6-Pyr)Gal.beta.1-4GlcNAc.beta.1-3Fuc;
<IMG>
e. analogues and derivatives thereof.
76. A therapeutic target comprising, at least one ligand on a cell surface
which binds to a
component of a purified extract from Microciona prolifera.
39

Description

CA 02505129 2005-05-05
WO 03/040368 PCT/CA02/01735
Pharmaceutical compositions of marine sponge Microciozza yrolifera
Field of the invention
The present invention provides pharmaceutical compositions and methods of use
for the
treatment of disorders with compounds originating from the marine sponge
Microciorza
pr~olifera.
Backyound of the Invention
Vascular, proliferative, autoimmune, and viral diseases are widespread,
devastating diseases
that affect millions of people worldwide. Such diseases include myasthenia
gravis, systemic
lupus erythematosus, atopic dermatitis, idiopathic pulmonary fibrosis,
multiple sclerosis,
diabetes, diabetic neuropathy, arthritis and other inflammatory joint
diseases, cancer and
metastasis, Alzheimer's disease, Parkinson's disease, amitrophic lateral
sclerosis, obesity,
osteoporosis, inflammatory dermatosis, inflammatory bowel disease, restenosis
following
vascular and coronary intervention, arteriosclerosis, wound healing disorders,
hepatitis B,
hepatitis C, HIV, Herpes simplex virus, genital warts, respiratory syncytial
virus,
rhinoviruses, adenoviruses, influenza and parainfluenza viruses and corona
viruses.
Currently, compounds for the treatment of these diseases are aimed towards
altering the
immune system, cell proliferation, cell adhesion and migration, and cytokine
levels or
activities.
There are several classes of molecules and disease processes that are common
to all these
diseases. These include increased expression of adhesion molecules, cytokines
and matrix
metalloproteinases, increased cell proliferation and migration, increased
inflammatory cell
activation and infiltration, increased angiogenesis, and increased tissue
destruction and
dysfunctional matrix remodeling. Experimental and clinical studies show that
tissue injury
leads to the hansition of cells to an activated state, followed by their
proliferation and
migration. Cellular migration and proliferation are facilitated by the release
of matrix
metalloproteinases that degrade the extracellular matrix. Certain components,
such as
hyaluronic acid, are especially detrimental since it functions as a cellular
signaling molecule,
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CA 02505129 2005-05-05
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eliciting the transition of cells to an activated state, with progression
along pathways leading
to cell proliferation and migration.
Sponges, the simplest multicellular organisms, are thought to have evolved
from their
unicellular ancestors by developing cell-recognition and adhesion mechanisms
to discriminate
against "non-self." Cellular adhesion of marine sponges is an event that
involves adherence of
extracellular proteoglycan-like molecules, otherwise known as aggregation
factors (Jarchow et
al., 2000).
The protein and carbohydrate components of sponge aggregation factors assemble
to form a
supramolecular complex very similar to classical proteoglycans. Although the
role of many
glycans in nature remains unclear, they have been implicated in many
biological processes,
including correct folding and secretion of proteins, and as receptor sites for
various
microorganisms and viruses (Spillmann et al., 1995)
The aggregation factor of Microciona sponge (MAF) is an adhesive proteoglycan-
like
molecule with a high carbohydrate content and a protein core. Fernandez-
Busquets et al. have
shown that a 35kDa protein is the basic unit of the core (MAFp3) with a
binding sequence
bearing a similarity to some other HA binding proteins (Fernandez-Busquets et
al., 1996).
There is growing evidence that carbohydrates, found on the surfaces of all
living cells, are
functional constituents in cell-cell interactions. Cell-cell interactions play
an important role in
the development, maintenance, and pathogenesis of tissues. They are highly
dynamic
processes that include migration, recognition, signaling, adhesion, and
finally attachment.
Cells on their pathway to a final location haveto pass and interact with
their substratum
formed of matrix and cell layers. Carbohydrates can, however, also lead to
diseases when they
are misused in pathological situations, by microorganisms or malignant cells,
for instance
(Heseley et al., 2001; Misevic et al., 1887).
Carbohydrates, which are the most prominent surface-exposed structures, must
play an
important role as recognition molecules in such processes. The rich
variability of
carbohydrate sequences that are presented by cell surface creates a refined
pattern of potential
attachment sites. Additionally, more interaction possibilities are created as
carbohydrates can
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CA 02505129 2005-05-05
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be branched while proteins cannot and oligosaccharide chains can be attached
to the protein
backbone in different densities and patterns.
Although the compounds for the treatment of the above listed diseases are
aimed towards
altering different pathological processes there is a need to develop improved
therapies for
restenosis, proliferative, autoimmune and viral diseases.
Summarx of the Invention
Within the context of the present invention, it should be noted that the above-
noted diseases
are deemed to be "treated" if the typical disease course is altered, slowed,
inhibited, or
prevented, for at least one symptom or sign of the disease.
The present invention provides pharmaceutical compositions for treating
restenosis and
autoimmune diseases, methods of treating restenosis and autoimmunes disease
with those
pharmaceutical compositions and use of the pharmaceutical compositions to
treat restenosis
and autoimmune diseases.
According to an aspect of the present invention, there is provided a
polypeptide comprising an
amino acid sequence selected from the group consisting of B-2 (SEQ ID NO: 2),
B-3 (SEQ ID
NO 3), B-6 (SEQ ID NO 4), B-9 (SEQ ID NO: 5), B-10 (SEQ ID NO: 6), and B-11
(SEQ ID
NO: 7).
According to another aspect of the present invention, there is provided a
pharmaceutical
composition comprising a polypeptide as disclosed above.
According to another aspect of the present invention, the pharmaceutical
composition as
described above is used for the treatment of restenosis, with or without the
addition of a
pharmaceutically acceptable carrier.
According to another aspect of the present invention, the pharmaceutical
composition as
described above is used for the treatment of an autoimmune disease, with or
without the
addition of a pharmaceutically acceptable carrier.
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According to a further aspect of the present invention, the said autoimmune
disease is selected
from the group consisting of multiple sclerosis, diabetes, and an inflammatory
joint disease
(such as arthritis).
According to another aspect of the present invention, the pharmaceutical
composition as
described above is used for the treatment of multiple sclerosis.
According to yet another aspect of the present invention there is provided an
antibody which
binds to a polypeptide comprising an amino acid sequence selected from the
group consisting
of B-1 (SEQ ID NO: 1), B-2 (SEQ ID NO: 2), B-3 (SEQ ID NO 3), B-6 (SEQ ID NO
4), B-9
(SEQ ID NO: 5), B-10 (SEQ ID NO: 6), B-11 (SEQ ID NO: 7) and analogues and
derivatives
thereof.
According to another aspect of the present invention, said antibody is a human
monoclonal
antibody or an Fab fragment of an antibody.
A further aspect of the present invention is immunization therapies and
vaccine compositions
comprising polypeptides listed above.
According to a further aspect of the present invention there is provided a
method for treating
restenosis comprising the step of administering to a patient a compound
selected from the
group consisting of (a) a polypeptide comprising an amino acid sequence
selected from the
group consisting of B-1 (SEQ ID NO: 1), B-2 (SEQ ID NO: 2), B-3 (SEQ ID NO 3),
B-6
(SEQ ID NO 4), B-9 (SEQ ID NO: 5), B-10 (SEQ ID NO: 6), B-11 (SEQ ID NO: 7),
and
analogues and derivatives thereof; and (b) an antibody to the polypeptide of
(a).
According to a further aspect of the present invention there is provided a
method for treating
autoimmune diseases (such as multiple sclerosis, diabetes, and inflammatory
joint disease
(such as arthritis)) comprising the step of administering to a patient a
compound selected from
the group consisting of (a) a polypeptide comprising an amino acid sequence
selected from
the group consisting of B-1 (SEQ ID NO: 1), B-2 (SEQ ID NO: 2), B-3 (SEQ ID NO
3), B-6
(SEQ ID NO 4), B-9 (SEQ ID NO: 5), B-10 (SEQ 1D NO: 6), B-11 (SEQ ID NO: 7),
and
analogues and derivatives thereof; and (b) an antibody to the polypeptide of
(a).
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According to a further aspect of the present invention there is provided a
method for treating
multiple sclerosis comprising the step of administering to a patient a
compound selected from
the group consisting of (a) a polypeptide comprising an amino acid sequence
selected from
the group consisting of B-1 (SEQ ID NO: 1), B-2 (SEQ ID NO: 2), B-3 (SEQ ID NO
3), B-6
(SEQ ID NO 4), B-9 (SEQ ID NO: 5), B-10 (SEQ ID NO: 6), B-11 (SEQ ID NO: 7),
and
analogues and derivatives thereof; and (b) an antibody to the polypeptide of
(a).
According to yet another aspect of the invention is a method for treating
restenosis
comprising the step of administering to a patient a compound selected from the
group
consisting of:
a. GIcNAc~i1-3Fuc;
b. Gal(31-4GlcNAc(31-3Fuc;
c. (4,6-Pyr)Gal(31-4GIcNAc~31-3Fuc;
d. GIcNAc(31-3Fuc
3503; and
e. analogues and derivatives thereof.
According to yet another aspect of the invention is a method for treating an
autoimmune
disease (such as multiple sclerosis, diabetes, and inflammatory joint disease
(such as
arthritis)) comprising the step of administering to a patient a compound
selected from the
group consisting of:
a. GIcNAc(31-3Fuc;
b. Gal(31-4GIcNAc(31-3Fuc;
c. (4,6-Pyr)Gal(31-4GlcNAc(31-3Fuc;

CA 02505129 2005-05-05
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d. GIcNAc(31-3Fuc
3503; and
e. analogues and derivatives thereof.
According to yet another aspect of the invention is a method for treating
multiple sclerosis
comprising the step of administering to a patient a compound selected from the
group
consisting of:
a. GIcNAc(31-3Fuc;
b. Gal(31-4GlcNAc(31-3Fuc;
c. (4,6-Pyr)Gal(31-4GlcNAc(31-3Fuc;
d. GIcNAc(31-3Fuc
3503; and
e. analogues and derivatives thereof.
According to yet another aspect of the present invention there is provided a
method for
treating restenosis comprising the step of administering to a patient a
pharmaceutical
composition comprising a purified extract from Microciofia pr-olifera.
According to yet another aspect of the present invention there is provided a
method for
treating autoimmune diseases (such as multiple sclerosis, diabetes, and
inflammatory joint
disease (such as arthritis)) comprising the step of administering to a patient
a pharmaceutical
composition comprising a purified extract from Microcioraa prolifera.
According to yet another aspect of the present invention there is provided a
method for
treating multiple sclerosis comprising the step of administering to a patient
a pharmaceutical
composition comprising a purified extract from Microciona prolifera.
6

CA 02505129 2005-05-05
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According to yet another aspect of the present invention, there is provided
the method of
treating restenosis comprising a step of administering to a patient at least
one compound
selected from a group consisting of (a) a polypeptide comprising an amino acid
sequence
selected from the group consisting of B-1 (SEQ ID NO: 1), B-2 (SEQ m NO: 2), B-
3 (SEQ
ID NO 3), B-6 (SEQ ID NO 4), B-9 (SEQ ID NO: 5), B-10 (SEQ ID NO: 6), B-11
(SEQ )D
NO: 7) and analogues and derivatives thereof; and (b) an antibody to said
polypeptide.
According to yet another aspect of the present invention, there is provided
the method of
treating autoimmune diseases (such as multiple sclerosis, diabetes, and
inflammatory joint
disease (such as arthritis)) comprising a step of administering to a patient
at least one
compound selected from a group consisting of (a) a polypeptide comprising an
amino acid
sequence selected from the group consisting of B-1 (SEQ ID NO: 1), B-2 (SEQ ID
NO: 2), B-
3 (SEQ ID NO 3), B-6 (SEQ ID NO 4), B-9 (SEQ ID NO: 5), B-10 (SEQ ID N0: 6), B-
11
(SEQ ID NO: 7) and analogues and derivatives thereof; and (b) an antibody to
said
polypeptide.
According to a further aspect of the present invention the treatment methods
described above
are administered wherein the dose is administered according to a regime
selected from a
group consisting of a single dose, multiple daily doses, multiple weekly doses
and multiple
monthly doses.
According to yet another aspect of the present invention, there is provided
the use of
polypeptide comprising an amino acid sequence selected from the group
consisting of B-1
(SEQ ID NO: 1), B-2 (SEQ ID NO: 2), B-3 (SEQ ID NO 3), B-6 (SEQ ID NO 4), B-9
(SEQ
ID NO: 5), B-10 (SEQ ID N0: 6), B-11 (SEQ ID NO: 7) and analogues and
derivatives
thereof for the treatment of restenosis.
According to yet another aspect of the present invention, there is provided
the use of
polypeptide comprising an amino acid sequence selected from the group
consisting of B-1
(SEQ ID NO: 1), B-2 (SEQ ID NO: 2), B-3 (SEQ ID NO 3), B-6 (SEQ ID NO 4), B-9
(SEQ
ID NO: 5), B-10 (SEQ ID NO: 6), B-11 (SEQ ID NO: 7) and analogues and
derivatives
thereof for the treatment of an autoimmune disease (such as multiple
sclerosis, diabetes, and
inflammatory joint disease (such as arthritis)).
7

CA 02505129 2005-05-05
WO 03/040368 PCT/CA02/01735
According to yet another aspect of the present invention, there is provided
the use of
polypeptide comprising an amino acid sequence selected from the group
consisting of B-1
(SEQ ID NO: 1), B-2 (SEQ ID NO: 2,), B-3 (SEQ ID NO 3), B-6 (SEQ ID NO 4), B-9
(SEQ
ID NO: 5), B-10 (SEQ )D NO: 6), B-11 (SEQ ID NO: 7) and analogues and
derivatives
thereof for the treatment of multiple sclerosis.
The invention further provides for the use of a purified extract from
Microciona prolifera or a
compound selected from the group consisting of:
a. GIcNAc(31-3Fuc;
b. Gal(31-4GlcNAc(31-3Fuc;
c. (4,6-Pyr)Gal(31-4GlcNAc(31-3Fuc;
d. GIcNAc(31-3Fuc
3503; and
e. analogues and derivatives thereof
for the treatment of restenosis.
The invention further provides for the use of a purified extract from
Microciona prolifera or a
compound selected from the group consisting of:
a. GIcNAc(31-3Fuc;
b. Gal(31-4GlcNAc(31-3Fuc;
c. (4,6-Pyr)Gal(31-4GlcNAc(31-3Fuc;
d. GIcNAc(31-3Fuc
3503; and
e. analogues and derivatives thereof
8

CA 02505129 2005-05-05
WO 03/040368 PCT/CA02/01735
for the treatment of an autoimmune disease (such as multiple sclerosis,
diabetes, and
inflammatory joint disease (such as arthritis)).
The invention further provides for the use of a purified extract from
Microciona prolifera or a
compound selected from the group consisting of:
a. GIcNAc(31-3Fuc;
b. Gal(31-4GlcNAc(31-3Fuc;
c. (4,6-Pyr)Gal(31-4GlcNAc(31-3Fuc;
d. GIcNAc(31-3Fuc
3503; and
e. analogues and derivatives thereof
for the treatment of multiple sclerosis.
According to a further aspect of the present invention there is provided the
use of an antibody
which binds to a polypeptide comprising an amino acid sequence selected from
the group
consisting of B-1 (SEQ ID NO: 1), B-2 (SEQ ID NO: 2), B-3 (SEQ ID NO 3), B-6
(SEQ ID
NO 4), B-9 (SEQ ID NO: 5), B-10 (SEQ ID NO: 6), B-11 (SEQ ID NO: 7) and
analogues and
derivatives thereof for the treatment of restenosis.
According to a further aspect of the present invention there is provided the
use of an antibody
which binds to a polypeptide comprising an amino acid sequence selected from
the group
consisting of B-1 (SEQ ID NO: 1), B-2 (SEQ ID NO: 2), B-3 (SEQ ID NO 3), B-6
(SEQ ID
NO 4), B-9 (SEQ ID NO: 5), B-10 (SEQ ID NO: 6), B-11 (SEQ ID NO: 7) and
analogues and
derivatives thereof for the treatment of an autoimmune disease (such as
multiple sclerosis,
diabetes, and inflammatory joint disease (such as arthritis)).
According to a further aspect of the present invention there is provided the
use of an antibody
which binds to a polypeptide comprising an amino acid sequence selected from
the group
consisting of B-1 (SEQ ID NO: 1), B-2 (SEQ ff~ NO: 2), B-3 (SEQ ID NO 3), B-6
(SEQ ID
9

CA 02505129 2005-05-05
WO 03/040368 PCT/CA02/01735
NO 4), B-9 (SEQ ID NO: 5), B-10 (SEQ ID NO: 6), B-11 (SEQ ID NO: 7) and
analogues and
derivatives thereof for the treatment of multiple sclerosis.
According to a further aspect of the invention there is provided a
pharmaceutical composition
for the treatment of restenosis comprising: a purified extract from Microciona
prolifera
having a peak at about 2.7kDa using MALDI spectroscopy; and a pharmaceutically
acceptable carrier.
According to a further aspect of the invention there is provided a
pharmaceutical composition
for the treatment of an autoimmune disease (such as multiple sclerosis,
diabetes, and
inflammatory joint disease (such as arthritis)) comprising: a purified extract
from Microciorza
prolifera having a peak at about 2.7kDa using MALDI spectroscopy; and a
pharmaceutically
acceptable carrier.
According to yet another aspect of the present invention, there is provided a
pharmaceutical
composition for the treatment of restenosis comprising: an extract from
Microciorza prolifera
obtained by collecting cuttings from the tips of branches of Microcioraa
prolifer-a;
a. washing the cuttings in cold calcium and magnesium free artificial seawater
(CMFASW), wherein said cuttings contain one or more MAF-containing cells
and a fibro-silacaceous support;
b. rotating the cuttings in CMFASW;
c. separating MAF-containing cells from the fibro-silacaceous support;
d. incubating and rotating the MAF-containing cells;
e. centrifuging the MAF-containing cells and retaining the supernatant;
f. adding EDTA and optionally a preservative to obtain a supernatant solution;
g. storing the supernatant solution at about 4 degrees for at least 120 hours;
h. dialyzing the supernatant to obtain the retenate; and

CA 02505129 2005-05-05
WO 03/040368 PCT/CA02/01735
i. lyophilizing the retentate to obtain the extract.
According to yet another aspect of the present invention, there is provided a
pharmaceutical
composition for the treatment of an autoimmune disease (such as multiple
sclerosis, diabetes,
and inflammatory joint disease (such as arthritis)) comprising: an extract
from Microciona
prolifera obtained by collecting cuttings from the tips of branches of
Microciona prolifera;
a. washing the cuttings in cold calcium and magnesium free artificial seawater
(CMFASW); wherein said cuttings contain one or more MAF-containing cells
and a fibro-silacaceous support;
b. rotating the cuttings in CMFASW;
c. separating MAF-containing cells from their fibro-silacaceous support;
d. incubating and rotating the MAF-containing cells;
e. centrifuging the MAF-containing cells and retaining the supernatant;
f, adding EDTA and optionally a preservative to obtain a supernatant solution;
g. storing the supernatant solution at about 4 degrees for at least 120 hours;
h. dialyzing the supernatant solution to obtain a retenate; and
i. lyophilizing the retentate to obtain the extract.
According to a further aspect of the present invention there is provided the
use of a
polypeptide comprising an amino acid sequence selected from the group
consisting of B-1
(SEQ ID NO: 1), B-2 (SEQ ID NO: 2), B-3 (SEQ ID NO 3), B-6 (SEQ ID NO 4), B-9
(SEQ
ID NO: 5), B-10 (SEQ ID NO: 6), and B-11 (SEQ ID NO: 7), analogues and
derivatives
thereof, purified extracts of Microciona prolifera, and carbohydrate compounds
selected from
the group consisting of
a. GIcNAc(31-3Fuc;
11

CA 02505129 2005-05-05
WO 03/040368 PCT/CA02/01735
b. Gal(31-4GlcNAc(31-3Fuc;
c. (4,6-Pyr)Gal(31-4GlcNAc(31-3Fuc;
d. GIcNAc(31-3Fuc
3503; and
e. analogues and derivatives thereof,
to further identify ligands on cell surface that may serve as potential
therapeutic targets.
According to yet another aspect of the present invention, there is provided a
therapeutic
targets comprising at least one ligand on a cell surface which bind to a
compound selected
from the group consisting of B-1 (SEQ ID NO: 1) B-2 (SEQ ll~ NO: 2) B-3 (SEQ
ID NO 3),
B-6 (SEQ ID NO 4), B-9 (SEQ ID NO: 5), B-10 (SEQ ID NO: 6), B-11 (SEQ ID NO:
7) and
analogues and derivatives thereof, a purified extract from Microciona
prolifera and a
compound selected from the group consisting of
a. GIcNAc(31-3Fuc;
b. Gal(31-4GIcNAc~31-3Fuc;
c. (4,6-Pyr)Gal(31-4GlcNAc(31-3Fuc;
d. GIcNAc(31-3Fuc
3503; and
e. analogues and derivatives thereof .
12

CA 02505129 2005-05-05
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Description of the Drawings
Figure 1 shows photographs demonstrating the inhibition of control and peptide
B-9-treated
(20 ~,g/ml) smooth muscle cell motility at 0 and 16 hours.
Figure 2 shows photographs demonstrating the inhibition of control and peptide
B-2-treated
(50 ~g/ml and 100 ~,g/ml) smooth muscle cell motility at 0 and 20 hours.
Figure 3 shows photographs demonstrating the inhibition of control and
carbohydrate RR-1-
treated (1 ~g/ml and 5 ~g/ml) smooth muscle cell motility at 0 and 20 hours.
Figure 4 shows photographs demonstrating the inhibition of control and Maf
2.7kDa purified
extract treated (10~.g/ml) smooth muscle cell motility at 0 and 20 hours.
Figure 5 is a graph showing the effect of B-1 peptide on clinical scores in a
mouse EAE
model.
Figure 6 is a graph showing the effect of B-9 peptide on clinical scores in a
mouse EAE
model.
Figure 7 is a MALDI spectroscopy performed in negative polarities showing a
predominant
peak at 2.7 kDa.
Figure 8 is a MALDI spectroscopy performed in positive polarities showing a
predominant
peak at 2.7 kDa
13

CA 02505129 2005-05-05
WO 03/040368 PCT/CA02/01735
Detailed description of the invention
Defazzitions
"CFA" is complete Freunds' adjuvant,
"Combination therapies" means therapeutic agent combined with other
therapeutic agent
"Compounds" mean agents for the treatment of diseases.
"Disease" means any abnormal condition of body functions or structure that is
considered to
be harmful to the affected individual and includes an illness or disorder
"EAE" means the experimental autoimmune encephalomyelitis mouse model; the
mouse
model for multiple sclerosis.
"MAF" is an adhesive proteoglycan from the marine sponge Macrociorzcz pz-
olifez-a.
"MALDI" (Mass Analysis Matrix Assisted Laser Desorption) / Flight (MALDI-TOF)
is a
mass analysis technique
"MBP" is myelin basic protein.
"PBS" (Phosphate buffer saline) is an injectable solution that serves as a
negative control
because it does not have any physiological or therapeutic effects.
"PLP" is an isoprotein proteolipid protein. PLP becomes predominant in the
adult.
"PTX "(pertussis toxin) is the major protein toxin produced by virulent
strains of Bordetella
pertussis, the organism that causes whooping cough. Pertussis toxin (PTX) is a
potent
ancillary adjuvant that primes macrophages used to elicit several different
autoimmune
diseases, including experimental allergic encephalomyelitis (EAE)
14

CA 02505129 2005-05-05
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Carbohydrates:
"RR1" is a non-sulfated disaccharide with the following structure:
GIcNAc(31-3Fuc
"RR2" is a sulfated disaccharide with the following structure:
GIcNAc(31-3Fuc
3503
"RR4" is a non-pyruvated trisaccharide with the following structure:
Gal(31-4GlcNAc(31-3Fuc
"RRS" is a pyruvated trisaccharide with the following structure:
(4,6-Pyr)Gal(31-4GlcNAc(31-3Fuc
Peptides
"Peptide B-1" is a 13 amino acid synthetic peptide, representing the putative
HA (hyaluronic
acid) binding sequence of MAF (marine sponge Microciona ~rolifera adhesive
proteoglycan),
having the following amino acid sequence:
SEQ ID NO: 1 Gly-Val-Ser-Val-Arg-Arg-Tyr-Arg-Asn-Arg-Val-Arg-Ile
(GVSVRRYRNRVRI)
"Peptide B-2" is a 15 amino acid synthetic peptide, derived from MAF, having
the following
amino acid sequence:
SEQ ID NO: 2 Val-Gly-Phe-Asp-Pro-Tyr-Asp-Tyr-Glu-Val-Asn-Glu-Ala-Asp-Gly
(VGFDPYDYEVNEADG)
"Peptide B-3" is a 10 amino acid synthetic peptide derived from MAF having the
following
amino acid sequence:
SEQ ID NO 3: Glu-Asp-Gln-Leu-Asp-Ala-Met-Asn-Leu-Ser
(EDQLDAMNLS)

CA 02505129 2005-05-05
WO 03/040368 PCT/CA02/01735
"Peptide B-6" is a 11 amino acid synthetic peptide derived from MAF having the
following
amino acid sequence:
SEQ ID NO 4: Gly-Ser-Gly-Ile-Gly-Asp-Glu-Pro-Thr-Thr-Ser
(GSGIGDEPTTS)
"Peptide B-9" is a 12 amino acid synthetic peptide derived from MAF having the
following
amino acid sequence:
SEQ ID NO 5: Arg-Phe-Val-Ile-Asn-Ile-Thr-Thr-Ser-Gly-Ser-Asp
(RFVINITTSGSD)
"Peptide B-10" is a 12 amino acid synthetic peptide derived from MAF having
the following
amino acid sequence:
SEQ ID NO 6: Cys-Phe-Leu-Thr-Pro-His-Gly-Val-Glu-Leu-His-Lys(CFLTPHGVELHK)
"Peptide B-11": is a 21 amino acid synthetic peptide derived from MAF having
the following
amino acid sequence:
SEQ ID NO 7: Asn-Gly-Ser-Ile-Gly-Pro-Arg-Gly-Leu-Pro-Gly-Val-Arg-Gly-Asp-Arg-
Gly-
Lys-Arg-Gly-Lys
(NGSIGPRGLPGVRGDRGKRGK)
The present invention is directed to novel peptides, carbohydrates and
extracts that originate
from the marine sponge Mi.crociofia prolifer-a and that have the ability to
inhibit cell
migration without affecting cell viability. These synthetic peptides are also
effective in
decreasing clinical signs of disease in the EAE mouse model for multiple
sclerosis. Therefore,
these peptides, carbohydrates and extracts have the potential to provide an
effective treatment
for restenosis, autoimmune, proliferative and viral diseases.
Peptides originatingLfrom MAFp3
The present invention includes synthetic peptide constructs B-1 (SEQ ID NO:
1), B-2 (SEQ
ID NO: 2), B-3 (SEQ ID NO 3), B-6 (SEQ ID NO 4), B-9 (SEQ ID NO: 5), B-10 (SEQ
ID
NO: 6), and B-11 (SEQ ID NO: 7) of a cloned peptide MAFp3 fragment (Fernandez-
Busquets
et al., 1996).
16

CA 02505129 2005-05-05
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The peptides of the invention may be prepared by chemical synthesis using
techniques well
known in the chemistry of proteins such as solid phase synthesis (Merrifield
et al., 1964) or
synthesis in homogenous solution (Houbemweyl, 1987). The peptides of the
invention may
also be produced by recombinant DNA technology by the methods known in the
art.
Antibodies may likewise be employed to treat or to prevent restenosis coronary
intervention,
autoimmune diseases, viral diseases and proliferative diseases. The invention
also provides
for antibodies which bind to polypeptides B-1 (SEQ ID NO: 1), B-2 (SEQ ID NO:
2), B-3
(SEQ ID NO 3), B-6 (SEQ ID NO 4), B-9 (SEQ ID NO: 5), B-10 (SEQ ID NO: 6), and
B-11
(SEQ ID NO: 7). The antibodies are generated using methods well known to those
in the art.
One of ordinary skill in the art will appreciate that a variety of alternative
techniques for
generating antibodies exist. In this regard, the following U.S. patents teach
a variety of these
methodologies and are thus incorporated herein by reference: U.S. Patent Nos.
5,840,479;
5,770,380; 5,204,244; 5,482,856; 5,849,288; 5,780,225; 5,395,750; 5,225,539;
5,110,833;
5,693,762; 5,693,761; 5,693,762; 5,698,435; and 5,328,834.
Once suitable antibodies have been obtained, they may be isolated or purified
by many
techniques well known to those of ordinary skill in the art (see Antibodies: A
Laboratory
Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988).
Suitable
techniques include peptide or protein affinity columns, HPLC (e.g., reversed
phase, size
exclusion, ion-exchange), purification on protein A or protein G columns, or
any combination
of these techniques.
The invention also includes vaccines the polypeptides listed above. Vaccines
can be made
using any method known in the art.
Carbohydrates derived from marine sponge M. prolifera
Carbohydrates of the present invention include:
GIcNAc(31-3Fuc (RR-1);
GIcNAc(31-3Fuc
17

CA 02505129 2005-05-05
WO 03/040368 PCT/CA02/01735
3503 (RR-2);
Gal(31-4GlcNAc~31-3Fuc (RR-4); and
(4,6- .Pyr)Gal(31-4GlcNAc(31-3Fuc (RR-5). These carbohydrates can be prepared
as described
in Spillmann et al., 1995.
Preparation of actiye extracts from Microcioraa pr-olifer-a
Purified extracts of Microciona prolifera, the red bread marine sponge, may be
obtained
through any method known in the art. Preferably, live specimens of Microciorva
prolifera are
collected. 1-2 mm cuttings from tips (growing portions) of branches on the
whole sponge are
removed, forming batches of approximately 300-500 grams. Several hundred
branches may
be present on a single adult sponge, thereby facilitating the collection of
large quantities of
cell-rich fragments. The cut pieces are first washed three times with cold
calcium and
magnesium free artificial seawater (CMFASW) prepared according to the MBL
formulary,
placed in a beaker with CMFASW and gently agitated on the rotating platform
for four hours
at 16 ° C. Aliquots of the soaked tips are then placed on CMF-washed
silk bolting cloth which
can be fashioned into a pouch in the gloved hand, and squeezed so as to
express MAF-
containing cells free of their fibro-silicaceous support into a second
container. Next, in order
to facilitate the release of intact MAF proteoglycan from MAF-containing
cells, the
suspension is then placed in a large Erlenmeyer flask and rotated at 120 rpm
for six hours at
16° C.
The suspension is then centrifuged in the cold at 2500 rpm for 20 min to
separate the MAF-
containing supernatant. Cold centrifugation is repeated on the sponge cell-
free supernatant
fluid at a speed of 9000 RPM for 30 min so as remove fine particulate matter,
such as
bacteria, spores etc. Thereafter, EDTA is added at a concentration of 1 mM
together with
sodium azide at 1/2000 as a preservative. The batch of crude MAF extract is
then maintained
at 4°C for at least 120 hr before any further processing. The
combination of calcium depletion
and EDTA chelation generates low molecular fragments from intact high
molecular weight
MAF whose carbohydrate subunits are linked by calcium.
Volume reduction may be performed by any method known to those skilled in the
art.
Preferably Spectra lPor dialysis tubing (Fisher catalog no. 08-671-26, 1000
MWCO) is heated
18

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in 1 mM EDTA, profusely rinsed with glass distilled water, and then are filled
and embedded
in polyethylene glycol (PEG- J,T. Baker catalog no. U 222-09) according to the
following
scheme. A shallow metal pan is lined with absorbent paper. The MAF-filled and
clamped
tubing is then placed in the pan in a snake-like configuration. Dry PEG powder
is added so as
to cover the tubing.
After the volume has been substantially reduced, preferably by 85-
90°Io, the concentrated
extract is centrifuged for 60 min at 18,600 rpm. The supernatant is then
divided into two
portions, each placed in suitably prepared Spectra/Por dialysis tubing (Fisher
catalog no. 08-
750-3B - 1000 MWCO). Dialysis is then carried out against distilled water in
the cold room.
The dialysate water may be kept in motion by a sliming bar and a magnetic
stirring apparatus.
Preferably, the water is replaced daily for a period of six days in order to
achieve a final
diffusatehetentate ratio of 200. The retentate is then shell frozen and
lyophilized.
The dry product may then be further purified prior to analysis. Membrane
separation
preferably takes placeusing Spectra/Por (Fisher catalog no. 08-750-SC - 3500
MWCO). 0.6
g of dried product is dissolved in 10 ml glass distilled water and placed in
washed dialysis
tubing with a 3500 MWCO and anchored in a graduate cylinder containing 500 ml
of water,
and stored for 120 hr in the cold room. The diffusates are pooled, frozen on
dry ice, and
lyophilized.
Pharmaceutical compositions
The present invention provides for pharmaceutical compositions comprising
synthetic
peptides, carbohydrates, as well as extracts from the marine sponge
Mdcrocio~aa prolifera.
Specifically, the pharmaceutical composition contains synthetic MAFp3 peptides
B-2 (SEQ
ID NO: 2), B-3(SEQ ID NO: 3), B-6(SEQ ID N0:4),B-9(SEQ ID NO: 5),B-10(SEQ ID
NO:
6), and B-11(SEQ ID NO: 7), carbohydrates RR-l, RR-2, RR-4 and RR-5, their
analogues,
derivatives and conjugates and/or purified extracts from Microcio~ea
prolifera.
These compositions are useful for the treatment of a number of diseases,
including restenosis,
autoimmune, viral and proliferative diseases.
The pharmaceutical compositions of the invention preferably contain a
pharmaceutically
acceptable carrier or excipient suitable for rendering the compound or mixture
administrable
19

CA 02505129 2005-05-05
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orally as a tablet, capsule or pill, or parenterally, intravenously,
intradermally, intramuscularly
or subcutaneously, rectally, via inhalation or via buccal administration, or
transdermally. The
active ingredients may be admixed or compounded with any conventional,
pharmaceutically
acceptable carrier or excipient. It will be understood by those skilled in the
art that any mode
of administration, vehicle or carrier conventionally employed and which is
inert with respect
to the active agents may be utilized for preparing and administering the
pharmaceutical
compositions of the present invention. Illustrative of such methods, vehicles
and carriers are
those described, for example, in Remirzgtora's Phar-fnaceutical Sciences, 4th
ed. (1970), the
disclosure of which is incorporated herein by reference. Those skilled in the
art, having been
exposed to the principles of the invention, will experience no difficulty in
determining
suitable and appropriate vehicles, excipients and carriers or in compounding
the active
ingredients therewith to form the pharmaceutical compositions of the
invention.
The compositions of the invention may also be conjugated to transport
molecules, monoclonal
antibodies or transport modalities such as vesicles and micelles that
preferentially target
recipient cells.
The composition of the present invention may also be formulated to be
contained within, or,
adapted to be released by a surgical or medical device or implant, such as,
for example stems,
sutures, catheters, prosthesis and the like Specifically, the active compound
may be covalently
linked or mixed or encapsulated in microcapsules with either polymeric or non-
polymeric
formulations which may coat, embed or impregnate or otherwise contact a
medical device that
is commercially available or is in research and development phase such as an
implant, stmt,
stmt graft, vascular graft, indwelling catheter, sutures, catheter, prosthesis
and the like. In
other cases, the active compound may contact a medical device such as an
implant, stmt, stmt
graft, vascular graft, indwelling catheter, sutures, catheter, prosthesis and
the like without any
formulations. Carriers can be either commercially available or in research and
development
phase.
The compounds of the present invention in the described dosages are
administered orally,
intraperitoneally, subcutaneously, intra-muscularly, transdermally,
sublingually or
intravenously as is known in the art. For example, for oral administration the
pharmaceutical
composition can be prepared orally, for example, in the form of tablets,
troches, capsules,
elixirs, suspensions, syrups, wafers, chewing gum or the like prepared by
procedures known

CA 02505129 2005-05-05
WO 03/040368 PCT/CA02/01735
to those skilled in the art. The amount of active compound in such
therapeutically useful
compositions or preparations is such that a suitable dosage will be obtained.
The therapeutically effective amount of active agent to be included in the
pharmaceutical
composition of the invention depends, in each case, upon several factors,
e.g., the type, size
and condition of the patient to be treated, the intended mode of
administration, the capacity of
the patient to incorporate the intended dosage form, etc.
It can generally be stated that the peptide, carbohydrate and the principal
active compound
isolated from the extract, or analogues or derivatives thereof should be
preferably
administered in an amount of at least 0.1 ~g/kg per injectable dose and up to
50 mg/kg per
dose. In the case of a stmt, the therapeutic amount of principal active
compound to be
administered to the intimal or lumenal layer of arterial walls may range from
0.01 ~g to about
50 mg.
Example 1~ Effect of peptides carbohydrates and extract (2.7K) on smooth
muscle cell
mi~-ation- In vitro Assay of Biological Activity
The following in vitro assays were conducted to illustrate the effect of
peptides, carbohydrates
and extract (2.7K) on the smooth muscle cell migration. The cells used in the
following
example were rat aortic smooth muscle cells, A-10. The cells were seeded at
2.5x105
cells/well in 6 well plates using Dulbecco's Modified Eagle Medium (Gibco BRL
Cat. #
11885-084),10% FBS and 1% Antibiotic Antimycotic (Gibco BRL Cat. # 15240).
Confluency of cells after about 8 hrs was between 8090%. Cells were injured
with the single
edge cell scraper (one injury/well) then washed twice with PBS and treated
with peptides B-1
(SEQ ID NO:1), B-2(SEQ ID N0:2), B-3 (SEQ ID NO: 3), B-6, (SEQ ID NO: 4), B-
9(SEQ
ID NO:S), B-10(SEQ ID N0:6) AND B-11(SEQ ID NO: 7) at concentrations ranging
from
10~.g/ml ml to 100 ~g/ml. Images were taken using a XS modulation objective
(Zeiss,
Germany) attached to a Zeiss Axiovert 100 inverted microscope equipped with
Hoffman
Modulation contrast optical filters (Greenvale, NY).
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CA 02505129 2005-05-05
WO 03/040368 PCT/CA02/01735
The images were taken at zero time and following the incubation of smooth
muscle cells with
peptide, carbohydrates and extracts for a period of up to 24 hrs (Figures 1,
2, 3 and 4). The
100% inhibition of cell motility without toxic effects was achieved with the
peptide
concentrations ranging from 10 ~,g/ml to 100 ~.g/ml, whereas the 100%
inhibition with
carbohydrates and 2.7K purified MAF extract was achieved with the
concentrations ranging
from 2 to 25 ~g/ml. The 100% inhibition of cell motility data is summarized in
Tables 1 and
2.
Table 1. Inhibition of smooth muscle cell motility with MAFp3 peptides
MAFp3 100% Inhibition
of Cell
Peptides Motility
B-1 100 pg/ml
B-2 50-100
B-3 ~ 50
B-6 5075
B-9 20
B-10 100
B-11 75100
Table 2. Inhibition of smooth cell motility with MAF carbohydrates and MAF
2.7K extract
MAF ~ 100°! Inhibition
Oligosaccharides of Cell Motility
MAF-2.710 2---10 ~cglmL
RR-1 disaccharides 5N 10
RR-3 trisaccharides 25
The results of the assay demonstrate that the compounds of the invention are
efficient
inhibitors of smooth muscle cell migration in vitro and therefore believed
that they may serve
as an endogenous inhibitor of smooth muscle cell migration in the intimal
lesion after injury
as well as in prevention and treatment of proliferative, viral and autoimmune
diseases.
Increased smooth muscle cell migration following injury is one of the
predominant causes of
obstruction of the vascular system following stenting and other vascular
procedures.
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CA 02505129 2005-05-05
WO 03/040368 PCT/CA02/01735
Specifically, smooth muscle cell migration is a major cause of restenosis (For
a review, see
Schwartz, 1997, Raines, 2000).
Example 2' Effect of B-1 and B-9 peptides on EAE mouse model
The EAE mouse model is the standard animal model used for testing therapeutic
compounds
in the treatment of multiple sclerosis (MS). Acute EAE was induced by
immunization of 3
months old SJL/J female mice (Jackson Lab.; Bar Harbor, Me.) with the MBP
(myelin basic
protein) and PTX (pertussis toxin). Each animal received an s.c. injection at
tail base of 200
~g MBP in 0.1 ml of CFA and received an i.v. injection of 200 ng of PTX;
pertussis toxin was
injected again 48 hours later. Mice (4 animals per group) were treated with
the peptide B-1
(SEQ ID NO:1) or peptide B-9 (SEQ ID NO: 5) at a dose of 5 mg/kg daily;
injections starting
on the day of first immunization. Treatment was stopped at time of sacrifice.
Mice were
monitored daily from day 7 after immunization for clinical signs of EAE and
were scored on a
scale of 0 to 5. A score of 0 represented the absence of signs while a score
of 5 was given to
moribund animals.
A marked improvement of mean clinical score was observed by day 13 at both
groups treated
with the peptides. At day 15, the mean clinical score was 1.7 in PBS treated
mice compared to
0.6 in B-9 treated animals; whereas the mean clinical score was 1.5 in PBS
treated mice
compared to 0.7 in B-1 treated animals. Treatment of the EAE mice with B-1 and
B-9 peptide
showed significant attenuation of clinical signs of MS symptoms by improvement
in mean
clinical score and a delay in progression to disability. As shown in figures5
and 6 , the peptide
treatments were able to decrease the clinical scores up to 60% in comparison
to non-treated
animals.
Example 3' Preparation of active extracts from Microciona prolifera
Live specimens of Microciona prolifera, the red bread marine sponge, were
collected in the
Cape Cod area by personnel of the Marine Resources Center (MRC) at the Marine
Biological
Laboratory (MBL), Woods Hole, Massachusetts. The adhesive proteoglycan of
sponge,
MAF, was prepared from 1-2 mm cuttings from tips (growing portions) of
branches on the
whole sponge. Several hundred branches may be present on a single adult
sponge, thereby
facilitating the collection of large quantities of cell-rich fragments. A
single batch was
23

CA 02505129 2005-05-05
WO 03/040368 PCT/CA02/01735
prepared from the cuttings derived from five adult sponges, each weighing 300-
500 gm. The
cut pieces were first washed three times with cold calcium and magnesium free
artificial
seawater (CMFASW) prepared according to the MBL formulary. They were placed in
a
beaker with CMFASW and were gently agitated on the rotating platform for four
hours at 16 °
C. Aliquots of the soaked tips were then placed on CMF-washed silk bolting
cloth which can
be fashioned into a pouch in the gloved hand, and squeezed so as to express
MAF-containing
cells free of their fibro-silicaceous support into a second container. The
yield of cells was
ca.2-2.5 X 10~ cells per ml. The suspension was then placed in a large
Erlenmeyer flask and
rotated at 120 rpm for six hours at 16° C, a process, which facilitates
the release of intact
MAF proteoglycan from MAF-containing cells.
Cells were separated from the MAF-containing supernatant by centrifuging the
suspension in
the cold at 2500 rpm for 20 min. Cold centrifugation was repeated on the
sponge cell-free
supernatant fluid at a speed of 9000 RPM for 30 min so as remove fine
particulate matter,
such as bacteria, spores etc. Thereafter, EDTA was added at a concentration of
1 mM together
with sodium azide at 1/2000 as a preservative. The batch of crude MAF extract,
consisting of
2-2.5 liters was maintained at 4°C for at least 120 hr before any
further processing. The
combination of calcium depletion and EDTA chelation generates low molecular
fragments
from intact high molecular weight MAF whose carbohydrate subunits are linked
by calcium.
Volume reduction was carried out in Spectra /Por dialysis tubing (Fisher
catalog no. 08-671-
26, 1000 MWCO) which had been heated in 1 mM EDTA beforehand and profusely
rinsed
with glass distilled water. The filled dialysis tubes were embedded in
polyethylene glycol
(PEG- J,T. Baker catalog no. U 222-09) according to the following scheme. A
shallow metal
pan was lined with absorbent paper. The MAF-filled and clamped tubing was then
placed in
the pan in a snake-like configuration. Dry PEG powder was then added so as to
cover the
tubing. At 48 hr an 85-90% reduction in volume had occurred. The concentrated
brown-
colored extract was spun for 60 min at 18,600 rpm, a procedure that yielded a
dark brown
pellet, and a substantial reduction of color in the supernatant. This was
divided into two
portions, each placed in suitably prepared Spectra/Por dialysis tubing (Fisher
catalog no. 08-
750-3B - 1000 MWCO). Dialysis was carried out against distilled water in the
cold room.
The dialysate water was kept in motion by a stirring bar and a magnetic
stirring apparatus.
The water was replaced daily for a period of six days to achieve a final
diffusate/retentate
ratio of 200. The retentate was shell frozen and lyophilized.
24

CA 02505129 2005-05-05
WO 03/040368 PCT/CA02/01735
Membrane separation using SpectralPor (Fisher catalog no. 08-750-SC - 3500
MWCO). 0.6
g of dried product dissolved in 10 ml glass distilled water was placed in
washed dialysis
tubing with a 3500 MWCO and anchored in a graduate cylinder containing 500 ml
of water,
and stored for 120 hr in the cold room. The diffusates were pooled, frozen on
dry ice, and
lyophilized. On examination of this material using MALDI spectroscopy a
predominant peak
was detected at 2.7 kDa. The result was consistent when MALDI spectroscopy was
performed
in negative and positive polarities (Figure 7 and Figure 8).
Although the invention has been described with preferred embodiments, it is to
be understood
that modifications may be resorted to as will be apparent to those skilled in
the art. Such
modifications and variations are to be considered within the purview and scope
of the present
invention.
References
Fernandez-Busquets X, Kammerer RA and Burger MM. A 35kDa Protein Is the Basic
Unit of
the Core from the 2X10 4-kDa. Aggregation Factor Responsible for Species-
specific Cell
Adhesion in the Marine Sponge Micr-ociona prolifera. 1996. Journal of
Biological Chemistry
271(38): 23558-23565
Haseley SR, Vermeer HJ, Kamerling JP, Vliegenthart JF. Carbohydrate self-
recognition
mediates marine sponge cellular adhesion. 2001. Proceedings of National
Academy of
Sciences 98(16): 9419-9424
Houbemweyl, Methods of organic chemistry, ed. E. Wansch, Vol. 15 I and II,
Tricme,
Stuttgart, 1987.

CA 02505129 2005-05-05
WO 03/040368 PCT/CA02/01735
Jarchow J, Fritz J, Anselmetti D, Calabro A, Hascall VC, Gerosa D, Burger MM,
Fernandez-
Busquets X. Supramolecular Structure of a New Family of Circular Proteoglycans
Mediating
Cell Adhesion in Sponge. 2000. Journal of Structural Biology 132: 95-105
Merrifield, Journal of American Chemistry Association 85: 2149-2154, 1964
Misevic GN, Finne J, Burger MM. Involvement of carbohydrates as multiple low
affinity
interaction sites in the self-association of the aggregation factor from the
marine sponge
Microciona prolifera. 1987. Journal of Biological Chemistry 262(12):5870-7
Raines, EW. The extracellular matrix can regulate vascular cell migration,
proliferation and
survival. 2000. International Journal of Experimental Pathology 81:173-182
Schwartz, SM. Smooth Muscle Migration in Atherosclerosis and Restenosis. 1997.
Journal of
Clinical Investigation 99(12): 2814-2817
Spillmann D, Thomas-Oates JE, van Kuik JA, Vliegenthart JF, Misevic G, Burger
MM, Finne
J. Characterization of a Novel Sulfated Carbohydrate Unit Implicated in the
Carbohydrate-
Carbohydrate-mediated Cell Aggregation on the Marine Sponge Microciona
prolifera. 1995.
Journal of Biological Chemistry 270(10): 5089-5097
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